第442E号试验：体外皮肤致敏：针对皮肤致敏不良结果途径中树突状细胞激活关键事件的体外皮肤致过敏试验

OECD/OCDEYou are free to use this material subject to the terms and conditions available athttp://www.oecd.org/termsandconditions/OECD GUIDELINE FORTHETESTING OFCHEMICALSIn VitroSkin Sensitisation Assays Addressing theAdverse Outcome PathwayKey Eventon Activation of Dendritic CellsGENERAL INTRODUCTIONActivation of dendritic cells Key Event basedTest GuidelineA skin sensitiser refers to a substance that will lead to an allergic response following skin contactas defined by the United Nations Globally Harmonized System of Classification and Labelling of Chemicals(UN GHS) (1). There is general agreement on the key biological events underlying skin sensitisation. Thecurrent knowledge of the chemical and biological mechanisms associated with skin sensitisation has beensummarised as an Adverse Outcome Pathway (AOP) (2), starting with the molecular initiating eventthrough intermediate events to the adverse effect, namely allergic contact dermatitis. In this instance, themolecular initiating event (i.e. the first key event) is the covalent binding of electrophilic substances tonucleophilic centres in skin proteins. The second key event in this AOP takes place in the keratinocytesand includes inflammatory responses as well as changes in gene expression associated with specific cellsignalling pathways such as the antioxidant/electrophile response element (ARE)-dependent pathways.The third key event is the activation of dendritic cells (DC), typically assessed by expression of specific cellsurface markers,genomic transcripts,chemokines and cytokines. The fourth key event is T-cell activationand proliferation, which is indirectly assessed in the murine Local Lymph Node Assay (LLNA) (3).This Test Guideline (TG) describesin vitroassays that address mechanisms described under theKey Event on activation of dendritic cells of the AOP for skin sensitisation (2). The TG comprises testmethods to be used for supporting the discrimination between skin sensitisers and non-sensitisers inThe test methods described in this TG are:•Human Cell Line Activation test(h-CLAT)•U937 cell line activation Test (U-SENS™)•Interleukin-8 Reporter Gene Assay (IL-8 Luc assay)•Genomic Allergen Rapid Detection (GARD™) for assessment of skin sensitisers (GARD™skin) ©OECD, (2024)accordance with the UN GHS (1). 1.2. OECD/OCDE2Background and principles of the test methods included in the KeyEvent basedThe assessment of skin sensitisation has typically involved the use of laboratory animals. Theclassical methods that use guinea-pigs, the Guinea Pig Maximisation Test (GPMT) of Magnusson andKligman, and the Buehler Test (TG 406) (4), assess both the induction and elicitation phases of skinsensitisation. The murine tests, the LLNA (TG 429) (3) and its two non-radioactive modifications, LLNA:DA (TG 442 A) (5) and LLNA: BrdU-ELISA (TG 442 B) (6), all assess the induction responseexclusively,and have also gained acceptance, since they provide an advantage over the guinea pig tests in terms ofanimal welfare together with an objective measurement of the induction phase of skin sensitisation.Mechanistically-basedin chemicoandinvitrotest methods addressing the first key event (OECDTG 442C (7)), and second key event (OECD TG 442D (8)) of the skin sensitisation AOP have been adoptedfor contributing to the evaluation of the skin sensitisation hazard potential of chemicals.Skinsensitisers have been reported to induce the expression of cell membrane markers such asCD40, CD54, CD80, CD83, and CD86 in addition to induction of proinflammatory cytokines, such as IL-1βand TNF-α, and several chemokines including IL-8 (CXCL8) and CCL3 (9) (10) (11) (12), associated withDC activation (2). Test methods described in this TG either quantify the change in the expression of cellthe surface marker(s) CD54 and CD86, the cytokine IL-8, or a series of genes (genomic biomarkersignature) thatare associated with the process of activation of monocytes and DC following exposure toHowever, as DC activation represents only one key event of the skin sensitisation AOP (2) (13),information generated with test methods measuring markers of DC activation alone may not be sufficientas stand-alone methods to conclude on the presence or absence of skin sensitisation potential ofchemicals. Therefore data generated with the test methods described in this Test Guideline are proposedto support the discrimination between skin sensitisers (i.e. UN GHS Category 1) and non-sensitisers whenused within Integrated Approaches to Testing and Assessment (IATA), together with other relevantcomplementary information, e.g. derived from in vitro assays addressing other key events of the skinsensitisation AOP as well as non-testing methods, includingin silicomodelling and read-across fromchemical analogues (13). Examples of the use of data generated with these methods within DefinedApproaches, i.e. approaches standardised both in relation to the set of information sources used and inthe procedure applied tothe data to